DNA barcoding coupled with high resolution melting for rapid identification of Ardisia gigantifolia and its toxic adulterants

Abstract

Ardisia gigantifolia is a medicinal plant; its herbal medicinal product has played important roles in the ways of life of people in China. However, commercial products of this herbal medicine are frequently adulterated with Rhododendron molle root and Clerodendrum cyrtophyllum stem. In fact, R. molle has hepatotoxic effects which could pose health issues to the end user. In this study, we applied DNA barcoding coupled with high resolution melting (Bar-HRM) to distinguish A. gigantifolia and adulterant products. Two DNA barcode loci (ITS2 and psbA-trnH) were chosen for Bar-HRM analysis. We found that ITS2 and psbA-trnH regions contain some inter-species variability, which showed enough discrimination capability for Bar-HRM analysis. We applied the Bar-HRM method to test the method, and found that some products labelled as “A. gigantifolia” collected from local markets in China were identified to contain C. cyrtophyllum. Our study demonstrated that DNA barcoding coupled with high resolution melting can be used as a routine test to guarantee the quality of A. gigantifolia herbal products.

Keywords:

• Ardisia gigantifolia
• DNA barcoding
• ITS2 and psbA-trnH
• Bar-HRM
• Rapid identification

Introduction

Ardisia gigantifolia, an endangered medicinal plant belonging to the genus Ardisia, is distributed only in Guangxi, Guangdong, Fujian and Jiangxi provinces, China [1]. The dry root of this plant is used as an important traditional Chinese medicine to treat rheumatoid arthritis and inflammatory diseases in China for thousands of years as evidenced by ancient records [2]. It is also widely used as a remedy for bruises, sprains, blood stasis, tumors and chronic ulcer diseases. Recently, various types of bioactive ingredients have been isolated from this medicinal plant, including quinones, coumarins, saponins and so on. Furthermore, compounds isolated from this species have a range of bioactivities, for example anti-HIV, anti-inflammation, anti-tumor, anti-oxidation properties and antivirus activity [3]. However, with the market demand increasing and wild resources decreasing, the population of A. gigantifolia has been destroyed at a phenomenal rate, and regarded as a rare medicinal plant [4]. Because of its high market demand, the price of A. gigantifolia is more expensive than other related medicinal plants in markets. The high price has led to substantially adulterated samples by the intentional substitution with other cheaper plants. Market surveys identified Rhododendron molle root and Clerodendrum cyrtophyllum stem as the main medicinal materials used to adulterate commercial products of A. gigantifolia [5]. Recently, evidence that R. molle has hepatotoxic effects was reported [6–7]. However, identifying medicinal materials by morphological characters is complicated because they have similar morphology [5]. It is more difficult to discern medicinal materials when they are dried, cut, or pulverized. Therefore, the accurate identification of A. gigantifolia is inportant for authenticity and quality of Chinese medicinal herb, as well as safety and health for consumers.

With the development of molecular biology, DNA-based methods have been used to identify medicinal plants more accurately. Among these methods, DNA barcoding is a useful tool and has been widely applied to identify original species to guarantee food safety and study biodiversity [8–10]. In 2010, ITS2 was suggested as a universal barcoding locus for most medicinal plants [11]. Since then, the ITS2 locus has been widely used for medicinal plants identification [12–15]. Meanwhile, a chloroplast genome region psbA-trnH has also been proposed as a complementary marker for barcoding medicinal plants [16]. These studies demonstrated that DNA barcoding is conducive to addressing the difficulties of identifying herbal materials and provide a new taxonomic method for herbal plants.

High resolution melting (HRM) is a new method based on PCR. This technique could distinguish different genotypes by the different shapes of whole melting transitions (Tm) for different real-time PCR products. When the temperature increases, the DNA duplex is denatured, the intercalating DNA dye releases from the DNA double helix and the fluorescence changes. This technology is used for sequence matching, mutation scanning and genotyping polymorphisms, and is increasingly applied in medical research, such as cancer testing [17–18]. Recently, it is also used for authentication of raw materials and products of Chinese medicinal herbs, as well as for exact quantification of the proportion of adulterants in herbal products [18–19].

This study aimed to establish a combined method to distinguish the commercial A. gigantifolia product and its adulterants accurately using DNA barcoding coupled with HRM analysis. Two DNA barcode sequences (ITS2 and psbA-trnH) were used to examine their effectiveness as barcodes for Bar-HRM in the identification of A. gigantifolia. Finally, Bar-HRM was applied to evaluate different commercial herbal medicines labelled as “A. gigantifolia” species collected from the local market.

Materials and methods

Plant material

Forty-three authenticated samples which were collected from southern China were used to validate the Bar-HRM assay. These contained nineteen A. gigantifolia samples, twenty C. cyrtophyllum, and four R. molle samples. The morphological identification of these samples was completed by Shizhong Mao at Guangxi Institute of Botany, Chinese Academy of Sciences. Each voucher specimen was kept in the Herbarium of Guangxi Institute of Botany, Chinese Academy of Sciences. In addition to voucher samples, twelve commercial products of A. gigantifolia species were bought from local markets in China.

DNA isolation and HRM-PCR amplification

The plant materials were frozen with liquid nitrogen and then ground into a powder. For each sample, genomic DNA was extracted from 50 mg of powder by the Plant Genomic DNA Kit (Tiangen Biotech Co.). The DNA concentrations of samples were measured by a spectrophotometer (Qubit 3.0, Invitrogen Co., USA), and then the samples were diluted to 50 ng/μL for the next step.

The ITS2 region and the psbA-trnH region were selected for identity analysis because they have low intra-specific sequence variation but have sufficient variable sites to distinguish most closely related species [12,13,16]. Universal primers from these two regions (psbAF: 5′-GTT ATG CAT GAA CGT AAT GCT C-3′, trnHR: 5′-CGC GCA TGG ATT CAC AAT CC-3′ and ITS2F: 5′-ATG CGA TAC TTG GTG TGA AT-3′, ITS3R: 5′-GAC GCT TCT CCA GAC TAC AAT-3′) were used for HRM-PCR. The HRM-PCR reactions using both regions and the subsequent data analysis were carried out using to the method described by Li et al [20]. The only difference was that the annealing temperature for the ITS2 region was 56 °C, while for the psbA-trnH region was 55 °C.

PCR product sequencing and species identification

After HRM analysis, all PCR products were directly sequenced using the automated ABI 3730 XL sequencer (Applied Biosystems). Each sequence was assembled using CodonCode Aligner (CodonCode, USA), and then aligned using MEGA5.1 software [21]. Finally, all the sequences were submitted to an online DNA barcoding database of herbal materials identification (http://www.tcmbarcode.cn). This database provides a system for herbal species identification, and also provides barcoding sequences for their closely related species, adulterants and substitutes [22]. The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study.

Results

Analysis of ITS2 and psbA-trnH sequences

The sequences of ITS and psbA-trnH loci of A. gigantifolia and its adulterants were submitted to DNA Barcoding System for Identifying Herbal Medicine (http://www. tcmbarcode.cn) for species discrimination. The results showed that herbal materials could be identified using this system (Table 1). The success rates for PCR amplification and sequencing of all the samples were 100% for the two barcodes. In total, 86 sequences for 43 individual plants were analyzed in this study.

Table 1. Original information of plant materials used in this study.

No.	Species	Source	ITS2	psbA-trnH
1	A. gigantifolia 1	Zhaoqing, Guangdong	KT906070	KT906032
2	A. gigantifolia 2	Zhaoqing, Guangdong	KT906080	KT906043
3	A. gigantifolia 3	Zhaoqing, Guangdong	KT906082	KT906045
4	A. gigantifolia 4	Zhaoqing, Guangdong	KT906083	KT906046
5	A. gigantifolia 5	Zhaoqing, Guangdong	KT906084	KT906047
6	A. gigantifolia 6	Zhaoqing, Guangdong	KT906085	KT906048
7	A. gigantifolia 7	Laibin, Guangxi	KT906086	KT906049
8	A. gigantifolia 8	Guilin, Guangxi	KT906087	KT906050
9	A. gigantifolia 9	Guilin, Guangxi	KT906088	KT906051
10	A. gigantifolia 10	Guilin, Guangxi	KT906071	KT906033
11	A. gigantifolia 11	Guilin, Guangxi	KT906072	KT906034
12	A. gigantifolia 12	Guilin, Guangxi	KT906073	KT906035
13	A. gigantifolia 13	Hechi, Guangxi	KT906074	KT906036
14	A. gigantifolia 14	Hechi, Guangxi	KY783481	KT906037
15	A. gigantifolia 15	Hechi, Guangxi	KT906075	KT906038
16	A. gigantifolia 16	Baise, Guangxi	KT906076	KT906039
17	A. gigantifolia 17	Baise, Guangxi	KT906077	KT906040
18	A. gigantifolia 18	Liuzhou, Guangxi	KT906078	KT906041
19	A. gigantifolia 19	Liuzhou, Guangxi	KT906079	KT906042
20	A. gigantifolia 20	Liuzhou, Guangxi	KT906081	KT906044
21	C. cyrtophyllum 1	Guilin, Guangxi	KT906052	KT906013
22	C. cyrtophyllum 2	Guilin, Guangxi	KT906062	KT906024
23	C. cyrtophyllum 3	Guilin, Guangxi	KY783482	KT906025
24	C. cyrtophyllum 4	Guilin, Guangxi	KT906063	KT906026
25	C. cyrtophyllum 5	Guilin, Guangxi	KT906064	KT906027
26	C. cyrtophyllum 6	Guilin, Guangxi	KT906065	KT906028
27	C. cyrtophyllum 7	Guilin, Guangxi	KT906066	KT906029
28	C. cyrtophyllum 8	Guilin, Guangxi	KT906067	KT906030
29	C. cyrtophyllum 9	Guilin, Guangxi	KT906068	KT906031
30	C. cyrtophyllum 10	Guilin, Guangxi	KT906053	KT906014
31	C. cyrtophyllum 11	Guilin, Guangxi	KT906069	KT906015
32	C. cyrtophyllum 12	Guilin, Guangxi	KT906054	KT906016
33	C. cyrtophyllum 13	Guilin, Guangxi	KT906055	KT906017
34	C. cyrtophyllum 14	Guilin, Guangxi	KT906056	KT906018
35	C. cyrtophyllum 15	Guilin, Guangxi	KT906057	KT906019
36	C. cyrtophyllum 16	Guilin, Guangxi	KT906058	KT906020
37	C. cyrtophyllum 17	Guilin, Guangxi	KT906059	KT906021
38	C. cyrtophyllum 18	Guilin, Guangxi	KT906060	KT906022
39	C. cyrtophyllum 19	Zhaoqing, Guangdong	KT906061	KT906023
40	R. molle 1	Zhaoqing, Guangdong	KT906005	KT906009
41	R. molle 2	Zhaoqing, Guangdong	KT906006	KT906010
42	R. molle 3	Zhaoqing, Guangdong	KT906007	KT906011
43	R. molle 4	Zhaoqing, Guangdong	KT906008	KT906012

A. gigantifolia, Ardisia gigantifolia; C. cyrtophyllum, Clerodendrum cyrtophyllum; R. molle, Rhododendron molle..

The length of the ITS2 sequences varied from 217 bp to 230 bp. For the ITS2 matrix, the intraspecific distances ranged from 0 to 0.48% and the interspecific distances ranged from 44.75% to 59.04%. The length of psbA-trnH sequences varied from 450 bp to 528 bp. For the psbA-trnH matrix, the intraspecific distances ranged from 0 to 0.87% and the interspecific distances ranged from 19.07% to 28.61%). The results showed that the two barcodes have high interspecific divergence and were suitable for further HRM analysis (Figures 1 and 2). The information of sequences (ITS2 and psbA-trnH) are available at https://figshare.com/s/e124acf847252f93d9b4.

Figure 1. Alignment of ITS2 locus of sequencing products of three herbal species, alignment information can be accessed at https://figshare.com/s/e124acf847252f93d9b4.

Figure 2. Alignment of psbA-trnH locus of sequencing products of three herbal species, alignment information can be accessed at https://figshare.com/s/e124acf847252f93d9b4.

HRM analysis based on ITS2 and psbA-trnH sequences

Two different curves were plotted to assess the melting characteristics of the ITS2 amplicons of all voucher samples (Figure 3). The three species were easily distinguished by different melting profiles using barcode marker ITS2 (Figure 3A). In the difference-plot melt curves (Figure 3B), improved visualization and separation of variant melting curves of each species were displayed. By assigning A. gigantifolia as the reference genotype, three species were successfully genotyped. Finally, all the examined species were shown indeed to be different, while the mean A. gigantifolia curve was used as the baseline.

Figure 3. High resolution melting analysis of a fragment of ITS2. (A) Curves of three herbal species in a normalized melting curve graph. (B) Difference graph of the normalized melting curves using A. gigantifolia as baseline. Information can be accessed at https://figshare.com/s/e124acf847252f93d9b4.

Analysis of the normalized HRM curves based on the barcode locus psbA-trnH is shown in Figure 4. Three species could be successfully distinguished because of their different melting profile (Figure 4A). Furthermore, using the mean A. gigantifolia curve as the baseline, closer examination of the HRM difference curve revealed part of the curve sitting outside the 90% CI curve, and the result suggested that the three examined species were indeed different (Figure 4B). DNA sequencing also confirmed the results obtained by HRM. These results all demonstrated that psbA-trnH was a sufficient marker to distinguish A. gigantifolia from its toxic adulterants.

Figure 4. High resolution melting analysis of a fragment of psbA-trnH. (A) Curves of three herbal species in a normalized melting curve graph. (B) Difference graph of the normalized melting curves using A. gigantifolia as baseline. Information can be accessed at https://figshare.com/s/e124acf847252f93d9b4.

Species identification of commercial products from local markets

Twelve samples of A. gigantifolia commercial herbal products were collected from Chinese local markets, and then were used to identify their accurate ingredients by Bar-HRM analysis. When using ITS2 primers, out of twelve samples, two samples labelled as A. gigantifolia were identified as C. cyrtophyllum (Figure 5). Using psbA-trnH primers, the same result was obtained (Figure 6). Each sequence of A. gigantifolia commercial herbal product was also analyzed to assess the results of HRM analysis. The blast results were consistent with the results by Bar-HRM (Figure 2).

Figure 5. Melting profiles of commercial products based on ITS2 region. (A) Normalized fluorescence curves of high resolution melting; (B) Reference-corrected normalized fluorescence plot of HRM. Information can be accessed at https://figshare.com/s/e124acf847252f93d9b4.

Figure 6. Melting profiles of commercial products based on psbA-trnH region. (A) Normalized fluorescence curves of high resolution melting; (B) Reference-corrected normalized fluorescence plot of HRM. Information can be accessed at https://figshare.com/s/e124acf847252f93d9b4.

Discussion

DNA barcodes are widely used for detecting substitution and adulteration of Chinese herbal medicines. In systematic research, ITS2 has been proposed as the core DNA barcode, while psbA-trnH was proposed as a supplementary DNA barcode to authenticate medicinal plants. Many previous studies have validated the identification capabilities of ITS2 and psbA-trnH locus [12–16]. The major advantages of DNA barcoding is its high sensitivity, and specificity. However, this method requires a molecular laboratory, costly equipment, and DNA sequencing machine; this inherent limitation hinders the wider implementation of DNA barcoding in developing countries. Therefore, to develop a sequencing free method that is faster and more economical than DNA barcoding is urgent. Bar-HRM is such a method and its discrimination capability is based on the length or base polymorphism of the target DNA regions. Bar-HRM could be used to identify the original species of traditional herbal products, whether a sample was a single ingredient sample or contained adulterants. Furthermore, the complete analysis of this simple and reliable technology is conducted in a closed tube system, and prevents cross-contamination risk [23–25]. Previous studies showed that Bar-HRM method has a potential powerful ability in identification for some herbal medicines [26–29]. However, until now, HRM analysis has not been widely utilized for identification of herbal products [30–32].

In this work, two DNA barcode regions (ITS2 and psbA-trnH) were selected for the authentication of A. gigantifolia. Bar-HRM assays were developed for both ITS2 and psbA-trnH regions. The results revealed that Bar-HRM is a simple and fast approach suitable for species identification. After HRM analysis, the PCR product of each sample was directly sequenced and the results confirmed that the samples could be directly genotyped by using the melting curves. For the determination of commercial frauds, a total of twelve commercial products sold as A. gigantifolia were analyzed. Two of these products were found to contain the ingredient of C. cyrtophyllum. Although this test did not find that the A. gigantifolia commercial herbal products contain toxic R. molle species on the market, our result demonstrated that DNA barcoding coupled with high resolution melting can provide a reference for the quality control and management strategies of A. gigantifolia products, as well as applied to test other herbal products [33–35].

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

All data generated during this study are available at Figshare (https://doi.org/10.6084/m9.figshare.13042928).

Additional information

Funding

This work was supported by the Major Scientific and Technological Special Project for “Significant New Drugs Creation” under Grant (2014ZX09201021), and the project of improving the basic scientific research ability of young and middle-aged teachers in Guangxi Universities in 2020 (2020KY12031).